1. Field of the Invention
The invention is directed to novel, purified and isolated IL-1 eta polypeptides and fragments thereof, the polynucleotides encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and uses thereof.
2. Description of Related Art
Interleukin-1 (IL-1) is a member of a large group of cytokines whose primary function is to mediate immune and inflammatory responses. There are seven known IL-1 family members which include IL-1 alpha (IL-1xcex1), IL-1 beta (IL-1xcex2), IL-1 receptor antagonist (IL-1ra), IL-1 delta (IL-1xcex4), IL-1 epsilon (IL-1xcex5), IL-1 zeta (IL-1"xgr") and IL-18 (previously known as IGIF and sometimes IL-1 gamma). IL-1 that is secreted by macrophages is actually a mixture of mostly IL-1xcex2 and some IL-1xcex1 (Abbas et al., 1994). IL-1xcex1 and IL-1xcex2 which are first produced as 33 kD precursors that lack a signal sequence, are further processed by proteolytic cleavage to produce secreted active forms, each about 17 kD. Additionally, the 33 kD precursor of IL-1xcex1 is also active. Both forms of IL-1 are the products of two different genes located on chromosome 2. Although the two forms are less than 30 percent homologous to each other, they both bind to the same receptors and have similar activities.
IL-1ra a biologically inactive form of IL-1, is structurally homologous to IL-1 and binds to the same receptors. Additionally, IL-1ra is produced with a signal sequence which allows for efficient secretion into the extracellular region where it competitively competes with IL-1 (Abbas et al., 1994).
The IL-1 family of ligands binds to a family of two IL-1 receptors, which are members of the Ig superfamily. IL-1 receptors include the 80 kDa type I receptor (IL-1RI) and a 68 kDa type II receptor (IL-1RII). IL-1 ligands can also bind to a soluble proteolytic fragment of IL-1RII (sIL-1RII) (Colotta et al., 1993).
The major source of IL-1 is the activated macrophage or mononuclear phagocyte. Other cells that produce IL-1 include epithelial and endothelial cells (Abbas et al., 1994). IL-1 secretion from macrophages occurs after the macrophage encounters and ingests gram-negative bacteria. Such bacteria contain lipopolysaccharide (LPS) molecules, also known as endotoxin, in the bacterial cell wall. LPS molecules are the active components that stimulate macrophages to produce tumor necrosis factor (TNF) and IL-1. In this case, IL-1 is produced in response to LPS and TNF production. At low concentrations, LPS stimulates macrophages and activates B-cells and other host responses needed to eliminate the bacterial infection; however, at high concentrations, LPS can cause severe tissue damage, shock, and even death.
The biological functions of IL-1 include activating vascular endothelial cells and lymphocytes, local tissue destruction, and fever (Janeway et al., 1996). At low levels, IL-1 stimulates macrophages and vascular endothelial cells to produce IL-6, upregulates molecules on the surface of vascular endothelial cells to increase leukocyte adhesion, and indirectly activates inflammatory leukocytes by stimulating mononuclear phagocytes and other cells to produce certain chemokines that activate inflammatory leukocytes. Additionally, IL-1 is involved in other inflammatory responses such as induction of prostaglandins, nitric oxide synthetase, and metalloproteinases. These IL-1 functions are crucial during low level microbial infections. However, if the microbial infection escalates, IL-1 acts systemically by inducing fever, stimulating mononuclear phagocytes to produce IL-1 and IL-6, increasing the production of serum proteins from hepatocytes, and activating the coagulation system. Additionally, IL-1 does not cause hemorrhagic necrosis of tumors, suppress bone marrow stem cell division, and IL-1 is lethal to humans at high concentrations.
Given the important function of IL-1, there is a need in the art for additional members of the IL-1 ligand and IL-1 receptor families. In addition, in view of the continuing interest in protein research and the immune system, the discovery, identification, and roles of new proteins (such as the human IL-1 eta of the invention) and their inhibitors, are at the forefront of modern molecular biology and biochemistry. Despite the growing body of knowledge, there is still a need in the art for the identity and function of proteins involved in cellular and immune responses.
The invention aids in fulfilling these various needs in the art by providing isolated polynucleotides and polypeptides encoded by the polynucleotides for the novel IL-1 family ligand termed xe2x80x9cIL-1 eta.xe2x80x9d Thus, in one aspect, the invention is directed to isolated novel polynucleotide molecules of IL-1 eta comprising the nucleotide residues 112-585 of SEQ ID NO:1 and to the isolated polynucleotide molecules encoding the amino acid sequence of SEQ ID NO:2, as well as polynucleotide molecules complementary to these sequences.
Both single-stranded and double-stranded RNA and DNA molecules are encompassed by the invention, as well as polynucleotide molecules that hybridize to a denatured, double-stranded DNA comprising all or a portion of SEQ ID NO:1 and/or a DNA that encodes the amino acid sequences set forth in SEQ ID NO:2. Also encompassed are isolated polynucleotide molecules that are derived by in vitro mutagenesis of polynucleotide molecules comprising the coding region of SEQ ID NO:1, that are degenerate from polynucleotide molecules comprising the sequence of SEQ ID NO:1, and that are allelic variants of DNA of the invention. The invention also encompasses recombinant vectors that direct the expression of these polynucleotide molecules and host cells transformed or transfected with these vectors.
In addition, the invention encompasses methods of using the DNA noted above to identify DNA encoding proteins having activities associated with IL-1 family ligands and receptors.
In addition, these polynucleotides can be used to identify the human chromosomes with which the polynucleotides are associated. Thus, since the IL-1 eta polynucleotides map to chromosome 2, DNA encoding IL-1 eta polypeptides may be used to identify human chromosome 2. Accordingly, these polynucleotides may also be used to map genes on human chromosome 2; to identify genes associated with certain diseases, syndromes, or other human conditions associated with human chromosome 2; and to study cell signal transduction and the immune system.
The invention also encompasses the use of sense or antisense oligonucleotides from the polynucleotides of SEQ ID NO:1 to inhibit the expression of the respective polynucleotide encoded by the genes of the invention.
The invention also encompasses isolated polypeptides and fragments of IL-1 eta as encoded by these polynucleotide molecules, including soluble polypeptide portions of SEQ ID NO:2. The invention further encompasses methods for the production of these polypeptides, including culturing a host cell under conditions promoting expression and recovering the polypeptide from the culture medium. Especially, the expression of these polypeptides in bacteria, yeast, plant, insect, and animal cells is encompassed by the invention.
In general, the polypeptides of the invention can be used to study cellular processes such as immune regulation, cell proliferation, cell death, cell migration, cell-to-cell interaction, and inflammatory responses. In addition, these polypeptides can be used to identify proteins associated with IL-1 eta ligands.
In addition, the invention includes assays utilizing these polypeptides to screen for potential inhibitors of activity associated with polypeptide counter-structure molecules, and methods of using these polypeptides as therapeutic agents for the treatment of diseases mediated by polypeptide counter-structure molecules. Further, methods of using these polypeptides in the design of inhibitors (e.g., engineered receptors that act as inhibitors) thereof are also an aspect of the invention.
Further encompassed by this invention is the use of the IL-1 eta polynucleotide sequences, predicted amino acid sequences of the polypeptide or fragments thereof, or a combination of the predicted amino acid sequences of the polypeptide and fragments thereof for use in searching an electronic database to aid in the identification of sample polynucleotides and/or proteins.
Isolated polyclonal or monoclonal antibodies that bind to these polypeptides are also encompassed by the invention, in addition the use of these antibodies to aid in purifying the polypeptides of the invention.